Platelets are small, anuclear blood cells which are essential to hemostatic control and wound healing. Circulating platelets are fairly quiescent under normal conditions. However, when a blood vessel is torn or damaged, platelets are exposed to various factors that instigate complicated and interconnected cellular programs leading to blood coagulation and clot formation, which are reviewed in Mechanisms of Platelet Activation and Control, K. S. Authi, S. P. Watson, and V. V. Kakar (eds.) Plenum Press, 1993 (incorporated herein by reference). The activation of these cellular programs result in dramatic increases in membrane adhesive properties, platelet aggregation, and the release of vasoconstrictive and fibrinolytic factors. As a consequence, a clot forms at the site of trauma, plugging any breach in the vessel wall and providing a substrate for fibroblast invasion and repair.
The early events in the clotting process can be functionally separated into two primary components: adhesion and activation. Adhesion is the process of “sticking” platelets to the injured vascular wall, whereas activation initiates complex physiological changes inside the cell. Together, these two processes result in platelet agglutination and, ultimately, in the production of a mature clot.
Most steps in these processes depend on the interaction of extracellular ligands with specific receptors embedded in the platelet cell membrane. In vivo, the first visible change in platelet behavior is the adhesion of platelets to an area of denuded endothelium. The initial contact involves interaction of the platelet glycoprotein complex GPIb-V-IX with von Willebrand factor (vWf) bound to the exposed epithelium. This interaction appears to be a reversible process which results in the “rolling” of platelets along the vessel wall. Although the vWf interaction does not completely immobilize circulating platelets, it is essential to platelet adherence under high blood flow conditions. Subsequent irreversible binding of glycoprotein GPIa-IIa (also known as integrin α2β1) to endothelial collagen stabilizes the vWf interaction event, firmly anchoring the platelet to the vessel wall. Unlike vWf, collagen adhesion appears to be a slower process and is effective only under low flow conditions, or after platelets have been partially arrested by vWf interactions. In addition,GPIa-IIa binding induces the flattening (spreading) of platelet against the vessel wall. Spreading promotes the binding of other subendothelial adhesion factors including fibronectin, vitronectin and thrombospondin. These post-spreading interactions further stabilize the adhesion of platelets to the vessel wall.
GPIa-IIa-dependent spreading represents one of the earliest manifestations of the activation process. Stimulation of GPIa-IIa and other collagen receptors induces a host of physiological changes. Among these are altered cell surface adhesion properties that result in platelet-platelet aggregation, and the secretion of various bioactive compounds. These compounds include the vasoconstrictor, epinephrine, and proclotting factors, which activate thrombin and lead to polymerization of fibrinogen into the fibrin threads of a mature clot. In addition, activated platelets release ADP and thromboxane A2 (TXA2). These powerful thrombogenic factors amplify the initial activation signal, recruiting additional platelets into the activated state.
In addition to GPIa-IIa, at least two other collagen receptors are expressed on the platelet cell surface, namely, GPIV (CD36), and GPVI. Recent evidence suggests that both of these receptors contribute to platelet activation. Nevertheless, roughly 3 to 10% of the Japanese population lack GPIV and these individuals do not appear to have any hemostatic abnormality. In fact, it has been speculated that these individuals may be protected against thrombotic ailments. In contrast, the substantially more rare individuals lacking either GPIa-IIa or GPVI exhibit prolonged bleeding times. GPIa-IIa deficiencies generally lead to more severe bleeding disorders than those of GPVI etiology. Nevertheless, these patients rarely present the life threatening hemophilias such as that seen in individuals lacking von Willibrand factor.
Observations of human variants, along with recent in vitro data, suggest that the three collagen receptors act in concert to mediate collagen-platelet interactions. In vitro, for instance, it is now possible to block the activity of each collagen receptor with antibodies specific for the collagen receptor sites. Individually, each antibody partially inhibits platelet adhesion to collagen and pairwise combinations of antibodies are significantly more inhibitory, particularly when GPIa-IIa and GPVI are inhibited simultaneously. Moreover, these studies demonstrate that GPIV, GPIa-IIa, and GPVI contribute to thrombosis through two distinct pathways, mechanistically distinguishable by the requirement for divalent metal cations.
Biochemical and sequence information indicates that GIPIa-IIa is a cation-dependent integrin-type receptor. In contrast, biochemical studies reveal that GPIV and GPVI do not require divalent metal cations and are thus non-integrin type. Of the non-integrin class, observations of human subjects clearly suggest that GPVI is more important than GPIV in the primary adhesion process. Indeed, in vitro experiments where GPIa-IIa function is blocked by chelating divalant cations, antibodies directed against GPVI completely abolish collagen-platelet interaction.
GPVI was first identified about 30 years ago by isoelectric focusing and electrophoresis. Until recently, its function was completely undefined and it was known merely as a platelet glycoprotein with a molecular mass of approximately 62 kDa under reducing condition. However, beginning around 1987, Dr. Minoru Okuma and associates examined several patients with a form of thrombocytopenic purpura, a bleeding/bruising syndrome characterized by accelerated platelet destruction and decreased numbers of circulating platelets. The platelets in some of Dr. Okuma's patients aggregated normally in response to most agonists, including ADP, thrombin, Ristocetin, and calcium ionophore (A23187) but were markedly unresponsive to collagen. Moreover, these platelets were found to have reduced amounts, or even totally lack, the 62 kDa glycoprotein. Sugiyama et al., Blood 69:1712–20 (1987); Moroi et al., J. Clin. Invest. 84:1440–45 (1989); Ryo et al., Am. J. Hematol. 39:25–31 (1992); and Arai et al., Brit. J. Haematol. 89:124–130 (1995).
The key reagent in the early studies of GPVI function came from the one of Dr. Okuma's thrombocytopenic purpura patients. This patient presented with massive, unexplained bleeding and was treated by transfusion with HLA-matched platelets. Subsequent detailed examination of the patient's blood revealed a total lack of GPVI. Most surprisingly, because this patient totally lacked GPVI, her immune system had identified the GPVI molecules on the transfused platelets as foreign antigens and produced polyclonal antibodies against GPVI. Sugiyama et al., Blood 69:1712–20 (1987).
A naturally occurring antibody is composed of two identical binding sites, specific for a single antigenic epitope. The two antigen-specific portions are linked by a common stem, or Fc domain, to form a complex capable of binding to two identical antigen molecules. Moreover, the divalent nature of the antibody, in conjunction with aggregatory properties of the Fc domain, allow cross-linking and aggregation of many specific antigen molecules. Dr. Okuma found that the divalent antibodies from the patient's serum caused a massive aggregation response when mixed with normal platelets. Conversely, when the antigen-specific domains are rendered monovalent by enzymatic removal of linking Fc domain, the resulting Fab fragments completely abolished collagen-induced aggregation of normal platelets and inhibited platelet-collagen adhesion.
Dr. Okuma has graciously made this rare serum available to the scientific community. Unfortunately, the supply is limited, and the circumstances surrounding its discovery are virtually irreproducible. Although the Okuma serum made possible much of the research into the function of GPVI, and had long provided the sole method of identifying a protein as GPVI, it has recently been discovered that the lectin, convulixin, specifically binds to GPVI with high affinity and can can be labeled as probe to identify the GPVI protein. (Francishetti et al., Toxicon 35:1217–28 (1997); Polgar et al., J. Biol. Chem. 272(24):13576–83 (1997); and Jandrot-Perrus et al., J. Biol. Chem. 272(2):27035–41 (1997) (which are both incorporated herein by reference.) Convulxin is a venom component from the tropical rattlesnake Crotalus durissus terrificus. In its native, divalent form, convuixin is a potent inducer of platelet aggregation and secretion of proaggregatory and proclotting factors. The divalent nature of convulxin is critical to the aggregatory effect. Although the underlying physiology of the reaction is unclear, individual convulxin subunits still bind to GPVI, but inhibits, rather than induces aggregation. It has been suggested that monovalent convuixin blocks the transmission of collagen-induced signals to the interior of the cell.
Recent evidence suggests that GPVI may be associated in the cell membrane with Fc receptor y (FcyRIIa). It is currently believed that collagen binding to GPVI induces tyrosine phosphorylation of FcyRII. Phosphorylated FcyRII then activates the Syk kinase, ultimately leading to a cascade of intracellular events including phospho-activation of cSrc, protein kinase c, and phospholipase C-γ2. These events ultimately result in increased intercellular calcium levels and the secretion of proaggregatory and proclotting factors.
It is now accepted that GPVI is the principle receptor for collagen-induced platelet activation, and is a critical conduit for signal transduction. Ichinohe et al., J. Biol Chem. 270(47):28029–28036 (1995); Tsuji et al., J. Biol Chem. 272(28):23528–31 (1997). In contrast, the other major collagen receptor in platelets, GPIa-IIa, is primarily involved with the cation-dependent processes of spreading and cell-cell cohesion.
Yet, despite the availability of research tools to elucidate the general mechanisms of GPVI function, this protein has proven remarkably refractory to purification. As a consequence, it has been impossible to generate anti-GPVI antibodies by conventional means, or even to purify sufficient protein to obtain a partial amino acid sequence. Lacking these reagents, no one has been able to identify the GPVI nucleotide or protein sequence. Indeed, Dr. Okuma, himself, has been unable to obtain the GPVI sequence. The lack of sequence data has thus severely hampered structure-function studies limited the search for GPVI agonists and antagonists.
The need in the art for GPVI sequence information, and GPVI antagonists, in particular, is highlighted by the unfortunate fact that inappropriate platelet aggregation and clot formation is a major etiologic factor in a wide range of human diseases, most commonly, vascular diseases. Excessive platelet aggregation in arteries and veins contributes to atherosclerotic and arteriosclerotic plaques which reduce the flow of blood to sensitive tissues. Ultimately, this platelet-dependent buildup may manifest as acute myocardial infarct, chronic unstable angina, transient ischemia, stroke, peripheral vascular disease, arterial thrombosis, preeclampsia, pulmonary embolism, restenois, and various other conditions.
These conditions typically begin with an abnormal clot that develops in a blood vessel, called a thrombus. Once a clot has developed, continued flow of blood past the clot is likely to break it free from its attachment. Such freely flowing clots are known as emboli. Emboli generally travel through the circulation until trapped in a narrow point in the circulatory system. This occlusion may occur in the brain, lung or cornary arteries, resulting in pain, disability or death.
Intravascular clots may result from naturally-occuring sclerosis, septicemic shock, or physical damage to blood vessels. Indeed, the very invasive methods used to diagnose and treat vascular disease, (e.g. vascular grafts, exploratory and in-dwelling catheters, stents, shunts, and other devices) themselves, damage vessel walls. This can activate plateles, stimulate aggregation, and ultimatly lead to the formation of thrombi and emboli, further endangering the life and health of the patient. Thus, methods for controling or reducing platelet aggregation and clot formation has been a long-sought goal in managing these diseases.
As a result of this increasing understanding of the physiology of platelet aggregation and clot formation, the traditional antithrombotics, aspirin, heparin, and ticlopidine, are increasingly being replaced or supplemented with new agents. One agent of great interest blocks the function of another platelet bound receptor GPIIb-IIIa. GPIIb-IIIa is the major platelet-specific integrin. It is activated by common platelet agonists such as thrombin and histamine to bind fibrinogen and von Willebrand factor. This ligand binding promotes platelet aggregation and the resulting thrombogenic. cascade. Various therapeautics designed to block GPIIb-IIIa activation have focused on the specificity of the ligand-receptor interaction.
It has been determined that the GPIIb-IIIa binding site recognizes the amino acid sequence, arginine-glycine-aspartate (RGD), which is found in a number of thrombogenic activators including fibrinogen and von Willebrand factor. Consequently, blocking this site inhibits platelet aggregation by preventing the receptor-ligand interaction. Although only 9 of the 20 known integrins recognize the RGD sequence, a major focus in the art has been to block ligand binding to the platelet specific GPIIb-IIIa receptor with peptides containing RGD. Because the RGD peptides appear to have poor stability and a short half life, RGD peptide derivatives, and non-peptide RGD mimetics are currently under development. In addition, GPIIb-IIIa antagonistic drugs based on a number of snake venom proteins with high affinity for the receptor are also under development. Each of these approaches is reviewed in Coller et al., Thrombosis and Haemostasis 74(1):302–308 (1995); and Windsteffer et al., Fibrinolysis & Proteolysis 11 (suppl. 1):85–96 (1997).
Clinically, the most successful GPIIb-IIIa inhibitor to date has been the Fab fragment of the mouse/human chimeric antibody, 7E3 (generic name abciximab; marketed under the tradename ReoPro™ by Eli Lilly and Centocor), which apparently blocks ligand-receptor interactions, including the interaction of GPIIb-IIIa with fibrinogen. (Coller et al., Blood 66:1456–59 (1986)). 7E3 is a potent inhibitor of platelet function and shows promise in reducing ischemic events after angioplasty and other invasive vascular events. Unfortunately, blocking GPIIb-IIIa, as with abciximab, has been associated with profound thrombocytopenia and increased incidents of hemorrhagic complications, including intracranial bleeding. (Bailey et al., Cath. and Cardiovas. Diag. 42:181–84 (1997).
Consequently, there remains a need in the art for safe and efficacious inhibitors of specific platelet functions. Recent research has thus focused on the platelet-specific collagen receptors. As discussed above, GPIV is an unlikely candidate for study due to the apparently minor contribution this receptor makes towards platelet function in vivo. Of the remaining collagen receptors, it is noted that the bleeding disorders associated with GPIIb-IIIa deficiencies are generally more severe than those associated with the lack of GPVI. Therefore, it is expected that blocking GPVI functions would provide the safer clinical alternative. Furthermore, antagonists of GPVI would be preferred over GPIIb-IIIa antagonists because GPVI is more intimately associated with the activation cascade, whereas GPIIb-IIIa is more prominently associated with aggregation leading thrombosis. It is therefore likely that the selective inhibition of GPVI may inhibit thrombosis without affecting hemostatic plug formation, thus providing new clinical weapons against platelet-mediated disease.
The cloning and sequencing of the integrin GPIIb-IIIa provided powerful tools for the functional dissection of this molecule and allowed for the design of important new therapeutic drugs. However, because GPVI collagen receptor has thus far proven refractory to cloning and sequencing, the understanding of GPVI and the design of specific antagonists against GPVI have been severly hampered. Thus, there is a longstanding need in the art to elucidate the GPVI protein and DNA sequences, to use that knowledge to identify GPVI active sites, design agonists and antagonists for research and therapy, generate GPVI peptides and antibodies directed against those peptides, provide nucleic acid probes, enable the in vivo and ex vivo recombinant production of GPVI sequences, and methods for purification and use of GPVI-containing cells and molecules.